Etudes sur le mécanisme de remodelage des nucléosomes par ...

tel-00413908, version 1 - 7 Sep 2009
H2A.Bbd nucleosomes resulted in a partial increase in restriction enzyme accessibility and
base excision repair (Angelov et al., 2004; Menoni et al., 2007).
The aforementioned studies strongly indicate the importance of H2A docking domain and C-
terminal region in the process of nucleosome remodeling. In this work, we have tried to
further elucidate the role and the mechanistic aspects of involvement of these domains in
nucleosome remodeling by SWI/SNF and RSC, two of the best characterized ATP dependent
chromatin remodeling complexes from yeast.
IV.2 Results
IV.2.1 Nucleosome reconstitutions with H2A C-terminal deletion, chimeric and variant
proteins
In order to understand the role of H2A docking domain and C-terminal part in nucleosome
remodeling we first made serial deletion mutants using the X. laevis N-terminal HA-tagged
H2A protein as the parent clone. A chimeric protein H2A.ddBbd was constructed in which the
docking domain of H2A was replaced with the docking domain of H2A.Bbd (H2A.ddBbd).
As a control full length H2A and H2A.Bbd were also used. Alignment of human H2A.1 and
H2a.Bbd are shown in figure IV.1A. Truncation points in deletion mutants are indicated by
arrowheads (in red) above the H2A.1 sequence. All the proteins were bacterially expressed
and purified in denaturing conditions as described in materials and methods section. The
purity of the recombinant proteins was checked by 18% SDS-PAGE (Figure IV.1B). We next
checked if the mutant proteins could be reconstituted in nucleosomes. For this, nucleosome
reconstitutions were performed using salt dialysis method and replacing conventional H2A by
mutant proteins in the reconstitution mixtures containing all the four histones and a NotI
digested 601 DNA. This DNA fragment strongly positions nucleosomes at one end and is an
ideal substrate for DNase I based footprinting assays. All the mutants and variant H2A
proteins were efficiently reconstituted in the nucleosomes as shown in figure IV.1C. Under
the reconstitution conditions very little free DNA was observed (with the exception of Δ79
nucleosomes where the amount of free DNA was slightly higher). This evidences for good
incorporation of mutant histones and reconstitution of bona fide nucleosomes. Note that the
nucleosomes containing deletion mutants of H2A exhibit a slower migration in the gel and
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